Tanzer M M, Meagher R B
Department of Genetics, University of Georgia, Athens 30602-7223, USA.
Mol Cell Biol. 1995 Dec;15(12):6641-52. doi: 10.1128/MCB.15.12.6641.
The degradation of the soybean SRS4 mRNA, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase, yields a set of proximal (5' intact) and distal (3' intact) products both in vivo and in vitro. These products are generated by endonucleolytic cleavages that occur essentially in a random order, although some products are produced more rapidly than others. Comparison of sizes of products on Northern (RNA) blots showed that the combined sizes of pairs of proximal and distal products form contiguous full-length SRS4 mRNAs. When the 3' ends of the proximal products and the 5' ends of the distal products were mapped by S1 nuclease and primer extension assays, respectively, both sets of ends mapped to the same sequences within the SRS4 mRNA. A small in vitro-synthesized RNA fragment containing one cleavage site inhibited cleavage of all major sites, equivalently consistent with one enzymatic activity generating the endonucleolytic cleavage products. These products were rich in GU nucleotides, but no obvious consensus sequence was found among several cleavage sites. Preliminary evidence suggested that secondary structure could play a role in site selection. The structures of the 5' ends of the proximal products and the 3' ends of the distal products were examined. Proximal products were found with approximately equal frequency in both m7G cap(+) and m7G cap(-) fractions, suggesting that the endonucleolytic cleavage events occurred independently of the removal of the 5' cap structure. Distal products were distributed among fractions with poly(A) tails ranging from undetectable to greater than 100 nucleotides in length, suggesting that the endonucleolytic cleavage events occurred independently of poly(A) tail shortening. Together, these data support a stochastic endonuclease model in which an endonucleolytic cleavage event is the initial step in SRS4 mRNA degradation.
编码1,5-二磷酸核酮糖羧化酶小亚基的大豆SRS4 mRNA在体内和体外降解时，都会产生一组近端（5'端完整）和远端（3'端完整）产物。这些产物是由内切核酸酶切割产生的，切割基本上是随机发生的，尽管有些产物的产生速度比其他产物快。通过Northern（RNA）印迹比较产物大小表明，近端和远端产物对的组合大小形成连续的全长SRS4 mRNA。当分别通过S1核酸酶和引物延伸试验对近端产物的3'端和远端产物的5'端进行定位时，两组末端都定位于SRS4 mRNA内的相同序列。一个含有一个切割位点的体外合成小RNA片段同等程度地抑制了所有主要位点的切割，这与一种产生内切核酸酶切割产物的酶活性一致。这些产物富含GU核苷酸，但在几个切割位点之间未发现明显的共有序列。初步证据表明二级结构可能在位点选择中起作用。研究了近端产物的5'端结构和远端产物的3'端结构。在m7G帽（+）和m7G帽（-）组分中发现近端产物的频率大致相等，这表明内切核酸酶切割事件的发生与5'帽结构的去除无关。远端产物分布在具有长度从不可检测到大于100个核苷酸的多聚（A）尾的组分中，这表明内切核酸酶切割事件的发生与多聚（A）尾缩短无关。总之，这些数据支持一个随机内切核酸酶模型，其中内切核酸酶切割事件是SRS4 mRNA降解的第一步。
